Characterization of proteins often requires the ability to incorporate detectable groups—e.g., fluorochromes, chromophores, spin labels, radioisotopes, paramagnetic atoms, heavy atoms, haptens, crosslinking agents, and cleavage agents—at specific, defined sites. For proteins that do not contain pre-existing cysteine residues, site-specific labeling can be accomplished by use of site-directed mutagenesis to introduce a cysteine residue at the site of interest, followed by cysteine-specific chemical modification to incorporate the labeled probe. However, for proteins that contain pre-existing cysteine residues, site-specific labeling is difficult. Multiple strategies have been reported: (i) intein-mediated labeling (“expressed protein ligation”), (Muir, et al., Proc. Natl. Acad. Sci. USA, 95:6705-6710 (1998)); (ii) transglutaminase-mediated labeling (Sato et al., Biochem. 35:13072-13080 (1996)); (iii) oxidation-mediated labeling (Geoghegan, et al., Bioconj. Chem., 3:138-146 (1992)); (iv) transition-metal-chelate-mediated labeling (Kapanidis et al., J. Amer. Chem. Soc., 123:12123 (2001)); and (v) trivalent-arsenic-mediated labeling (see, for example, Griffin et al., Science 281:269-272, 1998; U.S. Pat. Nos. 6,008,378 and 6,451,569 B1 to Tsien et al.; and copending, commonly owned U.S. application Ser. No. 10/461,224). Strategies (i)-(iii) do not permit in situ labeling (i.e., direct labeling of proteins in cuvettes, gels, blots, or biological samples—without the need for a subsequent purification step) or in vivo labeling (i.e., direct labeling of proteins in living cells). Strategy (iv) does not permit labeling and analysis at subnanomolar concentrations. Strategy (v) is the most useful in that it permits both in situ and in vivo labeling. However, strategy (v) previously has been limited to site-specific labeling with single fluorescent probes, and “statistical” labeling with multiple fluorescent probes. For example, in the “statistical” multi-labeling approach, probes A and B are reacted simultaneouslly with a target molecule or set of target molecules having two peptide tags, A′ and B′, and three possible products are formed: A:A′/A:B′, B:A′/B:B′ and A:A′/B:B′. However, only product A:A′/B:B′ has the desired properties. It would be preferred to provide a labeling method wherein probe A specifically bound only to tag A′, and wherein probe B specifically bound only to tag B′, so as to yield a defined, labeled product having the most desirable properties.
There is a need for improved methods and compositions for protein labeling that permit in situ labeling, that permit in vivo labeling, that permit labeling and analysis at sub-nanomolar concentrations, and that encompass a wide range of detectable groups with different properties. There is also a need in the art to prepare defined mult-labeled products. In particular, there is a need to extend the strategy of using arsenic-mediated labeling to permit orthogonal molecule-specific, site-specific labeling of target molecules, such as proteins, with multiple labels.